1. Field of the Invention
This invention relates to a medicament for treating and preventing platelet activation or thrombosis in the presence of heparin- and platelet factor 4-complex reactive antibodies using a two-O desulfated heparin and to a method for treating.
2. The Prior Art
The drug heparin, discovered almost a century ago, is used even today to prevent coagulation of the blood. Its application ranges from prevention of deep vein thrombosis in medical and surgical patients at risk for venous thrombosis and subsequent pulmonary embolism, to full anticoagulation as treatment of patients suffering pulmonary embolism, myocardial infarction, or other thrombotic disorders, and full anticoagulation in patients undergoing intravascular catheterization procedures or cardiac surgery, so that thrombosis is prevented on catheters or heart-lung bypass machines. Recently, heparin has also been found to be useful to treat disorders of vascular proliferation or inflammation, and has been shown beneficial in a plethora of other diseases, including secondary hypoxic pulmonary hypertension, asthma, cystic fibrosis, inflammatory bowel disease, eczema, burns and glomerulonephritis. However, heparin has two important and serious side effects limiting its use.
The first of these is its major therapeutic indication: excessive bleeding from anticoagulation. While anticoagulation is a benefit in prevention or treatment of thrombotic diseases, this is a drawback if heparin is used to treat other diseases such as asthma where anticoagulation is not needed for therapeutic benefit, and may even pose additional risk to the patient. Untoward bleeding from anticoagulation is even the principal side effect when heparin is used for prevention or treatment of thrombotic disorders where anticoagulation is indicated. Fortunately, the side effect of bleeding is usually self-limited. With termination of heparin therapy and replacement of any blood lost from the vascular space, coagulation function and blood pressure are usually restored to normal in a short time, ending the period of risk.
A second side effect, heparin-induced thrombocytopenia, is less frequent but far more serious. This condition refers to the fall in blood platelet counts occurring in some patients who receive heparin therapy in any form. The condition has been extensively reviewed by several authors (Fabris F, Ahmad S, Cella G, Jeske W P, Walenga J M, Fareed J. Pathophysiology of heparin-induced thrombocytopenia. Clinical and diagnostic implications—a review. Archiv Pathol Lab Med 124:1657-1666, 2000; Arepally G, Cines D B. Pathogenesis of heparin-induced thrombocytopenia and thrombosis. Autoimmunity Rev 1:125-132, 2002; Warkentin T E, Greinacher A. Heparin-induced thrombocytopenia and cardiac surgery. Ann Thorac Surg 76:638-648, 2003; Warkentin T E. Heparin-induced thrombocytopenia: pathogenesis and management. Brit J Haematol 121:535-555, 2003; Chong G H. Heparin-induced thrombocytopenia. J Thromb Haemostas 1: 1471-1478, 2003).
Two types of heparin-induced thrombocytopenia (HIT) exist. Heparin-induced thrombocytopenia-1 (HIT-1) is characterized by a brief and asymptomatic fall in the platelet count to as low as 100×109/L. This condition resolves spontaneously on its own and does not require discontinuation of the drug. It is thought that this condition is caused by heparin-induced platelet clumping, no immune component of the disease has been identified, and complications of the condition are unusual.
The second type of heparin-induced thrombocytopenia is more deadly. Heparin-induced thrombocytopenia-II (HIT-2) has an immunologic cause and is characterized by a profound fall in the platelet count (>50%) often after the fifth day of heparin therapy. In contrast to HIT-1, in which complications are rare, HIT-2 is usually accompanied by major arterial, venous or microvascular thrombosis, with loss of organ function or limb perfusion. Untreated, the condition can result in death. More common with heparin from bovine lung (5% of patients) than with porcine intestinal heparin (1% of patients), the incidence of the disease has varied widely, depending on the type of heparin, route of administration or patient population.
Intravenous heparin is associated with an overall incidence of HIT-2 of about 1.7%, whereas the condition is rare with subcutaneous prophylactic administration to prevent deep vein thrombosis (Schmitt B P, Adelman B. Heparin-associated thrombocytopenia: a critical review and pooled analysis. Am J Med Sci 305:208-215, 1993). Use of low molecular weight but fully anticoagulant heparins such as enoxaparin or dalteparin are less likely to result in the syndrome, but HIT-2 has been reported with low molecular weight heparins. The only anticoagulant thought to be completely free of risk from HIT-2 induction is the recently approved synthetic pentasaccharide factor Xa inhibitor fondaparinux sodium. (Walenga J M, Jeske W P, Bara L, Samama M M, Fareed, J, State-of-the-art article, Biochemical and Pharmacologic rationale for the development of a Heparin pentasaccharide. Thromb Res 86(1):1-36 (1997)).
The pathogenesis of HIT-2 is centrally focused upon platelet factor 4 (PF4), a 70-amino acid (7.78 kD) platelet-specific chemokine that is stored in platelet a granules, where it is bound to the glycosaminoglycan chondroitin sulfate. When released, PF4 self-associates into a tetramer of approximately 31 kD. PF4 is highly basic (32 lysine and 12 arginine residues per tetramer), rendering it highly positively charged. Normal plasma levels of PF4 are low, on the order of 8 nmol/L). That PF4 released from platelets following platelet activation binds to the glycocalyx of endothelial cells as a reservoir. The infusion of heparin transiently increases PF4 levels 15 to 30 fold for several hours by displacing PF4 from the vascular endothelial interface.
Formation of the PF4-heparin complex occurs optimally at equivalent stoichiometric concentrations of PF4 and heparin. When administered as a therapeutic anticoagulant, heparin levels range from 0.2 to 0.4 IU/ml, or 100-200 nmol/L, higher than optimum concentrations for PF4-heparin complex formation. However, in patients such as those undergoing cardiac or hip surgery, in vivo activation of platelets occurs, releasing PF4 into the circulation and increasing circulating PF4 levels (to 75-100 nmol/L) toward the optimal concentrations for 1:1 stoichiometric complex formation. When heparin binds to PF4, it produces a conformational change in the protein, exposing antigenic epitopes to which an antibody binds.
The HIT antibody binds heparin-PF4 complexes with high affinity. This antibody-heparin-PF4 complex then binds to platelets by attachment of the antibody Fc domain to the platelet Fc receptor (FcγRIIa). This in turn crosslinks the Fc platelet receptors, inducing platelet activation, thromboxane synthesis and platelet aggregation. PF4 released from the activated, aggregating platelets complexes with additional extracellular heparin to form additional heparin-PF4 complexes which bind to the platelet surface and serve as additional sites for HIT antibody binding. This next wave of HIT antibody binding to platelet-localized heparin-PF4 complexes occurs through the antibody's Fab domain, leaving the Fc domain free to interact with the Fc receptors of adjacent platelets, cross-linking FcγRIIa receptors and inducing additional platelet activation and aggregation. In parallel, platelet activation also results in CD40 ligand/CD40 release and interaction, resulting in the induction of tissue factor expression on the surface of endothelial cells and macrophages. The generation of platelet microparticles when HIT antibodies are present that are highly procoagulant; the up regulation of the adhesion molecule P-selection in the platelet surface; and the induction of a pro-inflammatory state with HIT shows the involvement of neutrophils and monocytes with platelets activated by HIT antibodies as well as cytokine up regulation in the patients. (Walenga J M, Jeske W P, Prechel M M, Makhos M. Newer insights on the mechanism of heparin-induced thrombocytopenia. Semin Thromb Hemost 30(Suppl 1):57-67 (2004)). This compounds the hypercoagulable state by providing stimulus for initiation of the extrinsic coagulation cascade, and provides the back-drop for the thrombotic complications of the HIT-2 syndrome. Thrombocytopenia is caused by clearance of activated platelets and platelet aggregates by the reticuloendothelial system.
The clinical syndrome characterizing HIT-2 is distinguished by a substantial fall in the platelet count by usually more than 50% to a median nadir of about 55×109/L. The fall in platelets can be accompanied by development of venous thrombosis and pulmonary embolism, or, less commonly, arterial thrombosis involving the large lower-limb arteries. Thrombotic stroke and myocardial infarction occurs less often. Another feature of the syndrome is the appearance of skin lesions at heparin injection sites, ranging in appearance from erythematous plaques to frank skin necrosis. A quarter of patients develop an acute syndrome of fever, chills, respiratory distress, hypertension and global amnesia when they receive heparin intravenously at a time when circulating HIT-2 antibodies are present. Even disseminated intravascular coagulation may result. To prevent these complications, it is suggested that when HIT-2 is recognized, the precipitating heparinoid should be stopped and the patient fully anticoagulated with an alternative agent such as a direct thrombin inhibitor (lepirudin, argatroban or bivalirudin) or the synthetic pentasaccharide fondaparinux, which does not cross-react with HIT antibodies. Because the use of warfarin acutely in the setting of HIT-2 has been associated with development of microvascular thrombosis or skin necrosis, long term follow-up anticoagulation with warfarin is delayed until resolution of thrombocytopenia. This often necessitates prolonged hospitalization for administration of alternative anticoagulants such as the direct thrombin inhibitors.
The structural features of heparinoids that are associated with HIT-2 have been characterized in detail (Greinacher A, Alban S, Dummel V, Franz G, Mueller-Eckhardt C, Characterization of the structural requirements for a carbohydrate based anticoagulant with a reduced risk of inducing the immunological type of heparin-associated thrombocytopenia. Thromb Haemostas 74:886-892 (1995); Walenga J M, et al, supra (2004); Walenga J M, Koza M J, Lewis B E, Pifarré R. Relative heparin induced thrombocytopenic potential of low molecular weight heparins and new antithrombotic agents. Clin Appl Thromb Hemost. 2(Suppl 1):S21-S27 (1996); and Jeske W P, Jay A M, Haas S, Walenga J M. Heparin-induced thrombocytopenic potential of GAG and non-GAG-based antithrombotic agents. Clin Appl Thromb Hemost 5(Suppl 1):S56-S62 (1999)). With linear heparin-like carbohydrate sulfates, the risk of platelet activation in the presence of a HIT antibody and PF4 was critically dependent upon both the molecular weight of the polymer and its degree of sulfation (i.e., average number of sulfates per carbohydrate monomer). The critical degree of sulfation to form the HIT-reactive heparin-PF4 antigenic complex was found to lie between 0.6 and 1.20 (i.e., 0.6 to 1.2 sulfate groups per carbohydrate monomer). The tendency of a sulfated polysaccharide to form the HIT reactive heparin-PF4 antigenic complex, with subsequent platelet activation, was also governed by molecular weight. Increasing concentrations of heparin were required for complex formation as heparins with decreasing molecular weight down to 2.4 kD were studied. With saccharides below 2.4 kD, no complex formation was observed. HIT antibody activation was also not observed with the synthetic pentasaccharide fondaparinux, which weighs about 1.7 kD. The investigators concluded that only two strategies predictably reduced the risk of HIT-reactive heparin-PF4 complex formation: 1) reducing degree of sulfation to <0.6 sulfates per carbohydrate unit; or 2) decreasing the molecular weight of the polysaccharide to <2.4 kD.
A heparin-like compound that does not interact with PF4 to form HIT-antibody reactive complexes would offer major advantages over unfractionated or low molecular weight heparins currently available for therapeutic use. Although there is no clinical proof as an anticoagulant, the new pentasaccharide fondaparinux appears to have achieved that goal, since it does not activate platelets in the presence of HIT antibody (Greinacher A, et al., supra; Walenga J M, et al, supra (2004); Walenga J M, et al., supra (1996); and Jeske W P, et al., supra (1999)). However, while ideal as an anticoagulant agent, this small molecular weight heparin analog is fully anticoagulant, placing the treated patient at risk for excess bleeding if he has a bleeding diathesis or rent in the integrity of his vascular system. This is especially problematic in subjects with HIT who have also suffered gastrointestinal or central nervous system hemorrhage. Anticoagulation with fondaparinux or any agent is necessary in HIT to prevent potentially fatal arterial or venous thrombosis, but can be life threatening if the subject is also actively hemorrhaging. Moreover, even if the subject is not hemorrhaging, a low anticoagulant strategy for treating HIT would be far safer and more preferable than the currently available strategies which are all fully anticoagulant drugs and which include the direct thrombin inhibitors, argatroban and lepirudin.
The present invention accomplishes this objective. A 2-O desulfated heparin has been synthesized which is useful as an agent to inhibit inflammation such as ischemia-reperfusion injury of the heart from myocardial infarction. It is an advantage of the present invention that methods to produce this 2-O desulfated heparin (ODS heparin) in large quantities on a commercial scale have been provided. ODS heparin also has greatly reduced USP and anti-Xa anticoagulant activity, rendering it safer for use in anti-inflammatory doses and less likely to cause bleeding. The average molecular weight of 2-O desulfated heparin is 10.5 kD, and its approximate degree of sulfation is 1.0 (5 sulfate groups per pentasaccharide, see FIG. 1), placing it well within the risk range for HIT antibody interaction (Greinacher A, et al., supra). Surprisingly and in spite of size and degree of sulfation which would predict otherwise, ODS heparin does not cause platelet activation in the presence of known HIT-reactive antiserum at low or high concentrations. Thus, ODS heparin also constitutes a safer alternative to other anti-inflammatory heparins by presenting significantly reduced risk for HIT-2 associated thrombocytopenia and thrombosis.
Even more surprisingly, 2-O desulfated heparin is also useful to treat the HIT syndrome once established and reduce the activity of platelet aggregation from an antibody directed against the heparin-PF4 complex. Thus, the administration of 2-O desulfated heparin, which has greatly reduced anticoagulant activity and bleeding risk, could be used as a clinical treatment for HIT syndrome, eliminating the need for risky, fully anticoagulant treatments currently in use for HIT therapy.